Showing papers by "Howard Hughes Medical Institute published in 1989"

Altering the genome by homologous recombination.

Abstract: Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.

The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA.

Abstract: HUMAN immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of one of these nuclear regulatory proteins, termed Rev (formerly Art or Trs), induces the cytoplasmic expression of the incompletely spliced, late class of HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env1–6. Here, we provide evidence that this induction reflects the export from the cell nucleus to the cytoplasm of a pool of unspliced viral RNA constitutively expressed in the nucleus. The hypothesis that Rev acts on RNA transport, rather than splicing, is further supported by the observation that the cytoplasmic expression of a non-spliceable HIV-1 env gene sequence is also subject to Rev regulation. Here we show that this Rev response requires a specific target sequence which coincides with a complex RNA secondary structure present in the env gene. The response to Rev is fully maintained when this sequence is relocated to other exonic or intronic locations within env but is ablated by inversion. These results indicate that the HIV-1 rev gene product induces HIV-1 structural gene expression by activating the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species.

Interaction of Staphylococcus aureus toxin "superantigens" with human T cells.

Abstract: A modification of the polymerase chain reaction has been used to establish the fact that a collection of Staphylococcus aureus toxins are "superantigens," each of which interacts with the T-cell alpha beta receptor of human T cells by means of a specific set of V beta elements.

The molecular basis for duchenne versus becker muscular dystrophy: correlation of severity with type of deletion

Abstract: About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.

Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts

Abstract: An important corollary to the recent advances in our understanding of the primary cause of Duchenne muscular dystrophy, is the validation of genuine genetic homologues as animal models of the disease in which potential therapies can be tested. The persistent skeletal muscle necrosis that characterizes human Duchenne muscular dystrophy is also seen in the mdx mouse and is, in both, a consequence of a deficiency of dystrophin, probably within the muscle fibres themselves. As injected muscle precursor cells of one genotype can fuse with host muscle fibres of a different genotype and express the donor genes, we decided to test grafts of normal muscle precursor cells to see if they could induce synthesis of dystrophin in innately dystrophin-deficient mdx muscle fibres. We show that injected normal muscle precursor cells can fuse with pre-existing or regenerating mdx muscle fibres to render many of these fibres dystrophin-positive and so to partially or wholly rescue them from their biochemical defect.

Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells

Abstract: Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

Genetic imprinting suggested by maternal heterodisomy in non-deletion Prader-Willi syndrome

Abstract: Prader-Willi syndrome (PWS) is the most common form of dysmorphic genetic obesity associated with mental retardation. About 60% of cases have a cytological deletion of chromosome 15q11q13 (refs 2, 3). These deletions occur de novo exclusively on the paternal chromosome. By contrast, Angelman syndrome (AS) is a very different clinical disorder and is also associated with deletions of region 15q11q13 (refs 6-8), indistinguishable from those in PWS except that they occur de novo on the maternal chromosome. The parental origin of the affected chromosomes 15 in these disorders could, therefore, be a contributory factor in determining their clinical phenotypes. We have now used cloned DNA markers specific for the 15q11q13 subregion to determine the parental origin of chromosome 15 in PWS individuals not having cytogenetic deletions; these individuals account for almost all of the remaining 40% of PWS cases. Probands in two families displayed maternal uniparental disomy for chromosome 15q11q13. This is the first demonstration that maternal heterodisomy--the presence of two different chromosome 15s derived from the mother--can be associated with a human genetic disease. The absence of a paternal contribution of genes in region 15q11q13, as found in PWS deletion cases, rather than a mutation in a specific gene(s) in this region may result in expression of the clinical phenotype. Thus, we conclude that a gene or genes in region 15q11q13 must be inherited from each parent for normal human development.

Expression of a large family of POU-domain regulatory genes in mammalian brain development.

Abstract: A novel region referred to as the POU-domain is present in two tissue-specific transcription factors, Pit-1 and Oct-2, that activate expression of genes specifying pituitary and lymphocyte phenotypes. We report the identification of multiple new members of a large family of POU-domain genes expressed in adult brain, and document that all the known mammalian POU-domain genes, including Pit-1 and Oct-2, are expressed widely in the developing nervous system.

Vβ-specific stimulation of human T cells by staphylococcal toxins

Abstract: The staphylococcal toxins are responsible for a number of diseases in man and other animals. Many of them have also long been known to be powerful T cell stimulants. They do not, however, stimulate all T cells. On the contrary, each toxin reacts with human T cells bearing particular V beta sequences as part of their receptors for major histocompatibility complex protein-associated antigen. The specificity of these toxins for V beta s puts them in the recently described class of superantigens and may account for the differential sensitivity of different individuals to the toxic effects of these proteins.

Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector.

Abstract: A P-element vector has been constructed and used to generate lines of flies with single autosomal P-element insertions. The lines were analyzed in two ways: (1) the identification of cis-acting patterning information within the Drosophila genome, as revealed by a lacZ reporter gene within the P element, and (2) the isolation of lethal mutations. We examined 3768 independent lines for the expression of lacZ in embryos and looked among these lines for lethal mutations affecting embryonic neurogenesis. This type of screen appears to be an effective way to find new loci that may play a role in the development of the Drosophila nervous system.

Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation

Abstract: One action of cyclosporin A thought to be central to many of its immunosuppressive effects is its ability to inhibit the early events of T lymphocyte activation such as lymphokine gene transcription in response to signals initiated at the antigen receptor. Cyclosporin A was found to specifically inhibit the appearance of DNA binding activity of NF-AT, AP-3, and to a lesser extent NF-kappa B, nuclear proteins that appear to be important in the transcriptional activation of the genes for interleukin-2 and its receptor, as well as several other lymphokines. In addition, cyclosporin A abolished the ability of the NF-AT binding site to activate a linked promoter in transfected mitogen-stimulated T lymphocytes and in lymphocytes from transgenic mice. These results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.

hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.

Abstract: hsp82 is one of the most highly conserved and abundantly synthesized heat shock proteins of eucaryotic cells. The yeast Saccharomyces cerevisiae contains two closely related genes in the HSP82 gene family. HSC82 was expressed constitutively at a very high level and was moderately induced by high temperatures. HSP82 was expressed constitutively at a much lower level and was more strongly induced by heat. Site-directed disruption mutations were produced in both genes. Cells homozygous for both mutations did not grow at any temperature. Cells carrying other combinations of the HSP82 and HSC82 mutations grew well at 25 degrees C, but their ability to grow at higher temperatures varied with gene copy number. Thus, HSP82 and HSC82 constitute an essential gene family in yeast cells. Although the two proteins had different patterns of expression, they appeared to have equivalent functions; growth at higher temperatures required higher concentrations of either protein. Biochemical analysis of hsp82 from vertebrate cells suggests that the protein binds to a variety of other cellular proteins, keeping them inactive until they have reached their proper intracellular location or have received the proper activation signal. We speculate that the reason cells require higher concentrations of hsp82 or hsc82 for growth at higher temperatures is to maintain proper levels of complex formation with these other proteins.

Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.

Abstract: In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.

NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus

Abstract: A CENTRAL assumption about long-term potentiation in the hip-pocampus is that the two classes of glutamate-receptor ion channel, the N-methyl-D-aspartate (NMDA) and the kainate/quisqualate (non-NMDA) subtypes, are co-localized at individual excitatory synapses1,2. This assumption is important because of the perceived interplay between NMDA and non-NMDA receptors in the induc-tion and expression of long-term potentiation: the NMDA class, by virtue of its voltage-dependent channel block by magnesium3,4 and calcium permeability5,6, provides the trigger for the induction of long-term potentiation, whereas the actual enhancement of synaptic efficacy is thought to be provided by the non-NMDA class7,9. If both receptor subtypes are present at the one synapse, such cross-modulation could occur rapidly and locally through diffusible factors. By measuring miniature synaptic currents in cultured hippocampal neurons we show that the majority (∼70%) of the excitatory synapses on a postsynaptic cell possess both kinds of receptor, although to different extents. Of the remaining excita-tory synapses, ∼20% contain only the non-NMDA subtype and the rest possess only NMDA receptors. This finding provides direct evidence for co-localization of glutamate-receptor subtypes at individual synapses, and also points to the possibility that long-term potentiation might be differentially expressed at each synapse according to the mix of receptor subtypes at that synapse.

Alternative splicing in the control of gene expression

Abstract: In this review, we focus upon the mechanistic, functional, and evolutionary aspects of alternative splicing. The discussion of mechanisms attempts to be exhaustive, drawing not only upon direct experimental investigations of alternatively spliced genes, but also upon relevant themes derived from the study of constitutive splicing

The DNA binding domain of the rat liver nuclear protein, C/EBP, is bipartite

Abstract: C/EBP is a rat liver nuclear protein capable of sequence-specific interaction with DNA. The DNA sequences to which C/EBP binds in vitro have been implicated in the control of messenger RNA synthesis. It has therefore been predicted that C/EBP will play a role in regulating gene expression in mammalian cells. The region of the C/EBP polypeptide required for direct interaction with DNA has been identified and shown to bear amino acid sequence relatedness with the product of the myc, fos, and jun proto-oncogenes. The arrangement of these related amino acid sequences led to the prediction of a new structural motif, termed the "leucine zipper," that plays a role in facilitating sequence-specific interaction between protein and DNA. Experimental tests now provide support for the leucine zipper hypothesis.

Protein encoded by v-erbA functions as a thyroid-hormone receptor antagonist.

Abstract: The thyroid-hormone receptor can, in the absence of its ligand, suppress activity of a responsive promoter. Addition of thyroid hormone, however, results in the stimulation of expression. The oncogenic derivative of the thyroid-hormone receptor, v-erbA, acts as a constitutive repressor and, when coexpressed with the receptor, blocks activation by thyroid hormone. Thus, v-erbA may be the first example of a dominant negative oncogene.

Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation.

Abstract: A three-dimensional crystal structure of the biotin-binding core of streptavidin has been determined at 3.1-A resolution. The structure was analyzed from diffraction data measured at three wavelengths from a single crystal of the selenobiotinyl complex with streptavidin. Streptavidin is a tetramer with subunits arrayed in D2 symmetry. Each protomer is an 8-stranded beta-barrel with simple up-down topology. Biotin molecules are bound at one end of each barrel. This study demonstrates the effectiveness of multiwavelength anomalous diffraction (MAD) procedures for macromolecular crystallography and provides a basis for detailed study of biotin-avidin interactions.

Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion.

Abstract: Many Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients have a cytogenetic deletion of 15q11q13. While AS and PWS share a similar cytogenetic anomaly, they have very different clinical phenotypes. DNAs from 4 AS patients were examined using 5 chromosome 15q11q13-specific cloned DNA segments. With the present level of resolution, the molecular deletions between AS and those previously reported for PWS did not appear to differ. However, in contrast to the paternal inheritance of the deleted chromosome 15 observed in the majority of PWS patients, maternal inheritance of the deleted chromosome 15 was demonstrated in the AS patients by restriction fragment length polymorphisms (RFLPs).

Calcium/calmodulin-dependent protein kinase II.

Abstract: Publisher Summary This chapter discusses the presently known calcium (Ca 2+ )/calmodulin (CaM)-dependent protein kinases and some of their properties. To date, CaM kinase II is the only Ca 2+ )/CaM-dependent protein kinase known to phosphorylate a wide range of proteins and for this reason, it is sometimes referred to as the CaM-dependent multifunctional protein kinase or CaM-dependent multiprotein kinase. These properties, mainly established in vitro , together with the wide distribution of the kinase, suggest that CaM kinase II may be involved in the regulation of numerous physiological functions. The chapter summarizes the present knowledge of CaM kinase II with the particular emphasis on the molecular mechanisms involved in the regulation of kinase activity. It also reviews the literature concerning the putative physiological functions of the kinase. There is a great deal known about the in vitro properties of calmodulin (CaM) kinase II, both in terms of its substrate specificity and its regulation by CaM and autophosphorylation. Much of this characterization is based on the experiments performed with rat brain isozyme of CaM kinase II, although in the aspects examined to date isozymes of the kinase from other tissues appear to behave in a broadly similar manner in vitro . However, relatively little is known about the functions of the kinase in vivo . Investigation of the physiological role of the kinase in brain and other tissues will be a particularly exciting area for future work. The current knowledge of the in vitro properties and the availability of complimentary DNA (cDNA) clones will hopefully expedite this research.